Incomplete healing of bone defects in the craniofacial skeleton is common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, -4), promote healing in bone defects, but the proteins' short half-lives and rapid clearance by the bloodstream limit their utility. The main goal of our initial R01 project was the development of tissue engineering approaches, based on muscle-derived stem cells (MDSCs), to efficiently deliver osteogenic proteins and improve craniofacial bone healing. We would like to thank NIDCR for their support during the initial funding period. We met and exceeded all the key objectives in the original R01 application, and our results formed the basis for 24 papers and 52 abstracts. We demonstrated that MDSCs genetically engineered to express BMP2 and BMP4 differentiate toward an osteogenic lineage and can improve bone healing in calvarial and long bone defects. We also found that concomitant expression of vascular endothelial growth factor (VEGF) improves the bone healing observed after implantation of BMP2-and BMP4-expressing MDSCs. This DE013420 competitive renewal application outlines experiments designed to extend our initial findings and facilitate the development of clinical applications of MDSCs to improve bone healing. First, we will examine the effect of the sex and age of donor mice on the number and the osteogenic potential of MDSCs. We will then determine if muscle biopsy size and source, time of culturing, hormonal stimulation, or ex vivo cyclic mechanical strain influence the number or osteogenic potential of MDSCs and might enable us to counterbalance sex-related differences exhibited by MDSCs. This research is integral to the clinical applicability of MDSCs for autologous bone tissue engineering applications, which will necessitate the isolation of highly osteogenic cells from patients of different sexes and ages. Next/we will investigate ways to optimize bone formation and healing by using MDSC-based tissue engineering techniques, including genetic manipulation and injectable scaffolds. Finally, we will use the results from the first 2 aims to isolate and characterize the human equivalents of the mouse MDSCs with the highest osteogenic potential and optimize their use for bone regeneration and repair. The proposed study will provide important information regarding the basic biology of MDSCs and their use for bone healing and should foster the development of clinical treatments for osseous deficiencies. [unreadable] [unreadable] [unreadable]